CDK8磷酸化雌激素受体和乳腺癌内分泌治疗耐药性机制的研究

Estrogen receptor alpha (ER) is overexpressed in approximately 70% of breast cancer. ER plays an important role in mammary tumorigenesis and governs response of breast cancer to endocrine therapy. Upon estrogen binding, ER undergoes conformational changes and sequential posttranslational modifications, such as phosphorylation, acetylation, and ubiquitination. Molecules that are responsible for these modifications of ER have been intensively studied in last two decades; however, how these modifications are coordinated in cells after estrogen exposure is poorly understood. Our preliminary studies suggest that CDK8 (cyclin-dependent kinase 8) may function as an integrator of multiple signals that modulate ER protein. ..CDK8, identified as an oncoprotein, promotes the proliferation of melanoma and colon cancer cells. By interrogating a large cohort of breast cancer patient samples, we observed that an increased expression of CDK8 and CCNC (encoding CycC) genes predicts favorable outcome in survival in ER-positive breast cancer patients, and knockdown of either CDK8 or CycC expression reduces estrogen-dependent cell proliferation. These observations underscore the importance of CDK8-CycC in ER-dependent breast cancer progression. Using Drosophila as a genetic model system and discovery tool, we demonstrate that CDK8 and CycC play a conserved role in regulating EcR (ecdysteroid receptor) function. CDK8 and CycC mutants delay the larval-to-pupal transition, a process controlled by EcR, the major steroid hormone receptor identified in arthropods. Strikingly, CDK8-CycC also regulates a group of genes composing of Ubiquitin Proteasome Pathway. ..Based on these pilot studies, we hypothesize that CDK8 plays a conserved role in regulating the transcriptional activities of steroid hormone receptors through direct phosphorylation and subsequent ubiquitin/proteasome-mediated degradation of the receptors. Thus the objective of this proposal is to understand the clinical significance of CDK8-CycC in breast cancer, how CDK8 regulates the transcriptional activities of ER, and to explore the conserved mechanisms of how CDK8 regulates the nuclear receptor activity...Specifically, we propose to determine the role of CDK8-CycC in breast cancer progression and patients’ responsiveness to endocrine therapy. In addition, we will determine the molecular mechanisms by which CDK8 regulates ER-dependent functions in breast cancer cells, respectively. Given the importance of ER in human breast cancers, identification of CDK8-CycC as a novel regulator of these processes will advance our understanding of cancer progression. Establishing the molecular mechanisms for elevated ER activation in breast cancer could lead to new strategies to prevent and treat breast cancer.

乳腺癌是女性最常见的恶性肿瘤，约70%初发患者的肿瘤组织雌激素受体（ER）阳性。针对ER阳性肿瘤病人，内分泌治疗是术后药物治疗的基本手段，但耐药性的产生让很多患者无法长期受益。研究显示耐药性的产生和ER蛋白质的翻译后修饰密切相关。前期工作发现：ER被磷酸化、乙酰化和泛素化等修饰；CDK8 及其配体基因在至少一种类型乳腺癌中高表达；CDK8促进乳腺癌细胞增殖；CDK8结合并磷酸化ER；全基因组表达分析显示CDK8调控泛素化途径。我们推测CDK8通过磷酸化ER，阻碍其乙酰化，促进其泛素化介导的降解。本研究我们拟1）利用人乳腺癌样本探索CDK8磷酸化ER的表达规律及与内分泌治疗敏感性的关系；2）利用细胞及动物模型验证CDK8对ER功能的调节；3）研究CDK8磷酸化ER和乳腺癌细胞获得激素非依赖性增殖能力的关系。本研究将丰富乳腺癌内分泌治疗耐药性产生的理论基础，并为耐药性逆转提供潜在的分子靶点。

乳腺癌是女性最常见的恶性肿瘤，和大多数恶性肿瘤一样，尽管乳腺癌存在很强的异质性，其发生发展涉及大量信号分子的共同作用。临床上超过70%的初诊乳腺癌均为雌激素受体阳性型， 显示了雌激素及受体（ER）在乳腺癌发生发展中的重要地位。虽然其它亚型乳腺癌具有更高的死亡率，但基于ER阳性型占绝大多数，其预后极大影响着乳腺癌整体的治疗效果，而且这类乳腺癌对雌激素内分泌治疗产生的抗性问题一直是乳腺癌研究的重点方向之一。.在所有抗雌激素治疗的药物中，最重要的为它莫昔芬（tamoxifen）类药物，是近三十年来抗雌激素生物治疗的一线药物。临床统计发现，5年的辅助性它莫昔芬治疗可降低乳腺癌47%的复发率及26%的死亡率，半数以上的ER阳性乳腺癌对它莫昔芬治疗有效或保持肿瘤不再进展。然而有不部分ER阳性乳腺癌患者从发病开始就对它莫昔芬治疗无效（原发性耐药），并且几乎所有的最初治疗敏感的乳腺癌最终都会发展为内分泌治疗继发性耐药（acquired endocrine resistance）。这种原发和继发耐药性严重影响了抗雌激素治疗的长期应用，使得即便是ER阳性乳腺癌患者最终也会失去对抗雌激素治疗的反应。因此，抗雌激素内分泌治疗中最棘手的瓶颈问题就是耐药性的产生。.本课题组前期的工作涉及雌激素受体ER的表达和活动调控，作为乳腺癌细胞内重要的核受体，ER的活性调节是通过翻译后修饰及与一系列共调节因子的相互作用实现的。通过与配体结合，ER可通过磷酸化、乙酰化、苏素化和泛素化等方式在翻译后被加以修饰。我们和其他国际上多个实验室的研究显示这些修饰和细胞增殖信号和/或临床抗雌激素治疗耐药性的产生有密切关系。在本项研究中，我们首先利用人乳腺癌样本探索CDK8磷酸化ER的表达规律及与内分泌治疗敏感性的关系；接下来我们利用细胞模型验证CDK8对ER功能的调节，成功构建了细胞模型并对CDK8调节ER依赖型细胞增殖进行了研究；最后我们深入机制研究，明确CDK8磷酸化ER的位点，初步探讨了乳腺癌CDK8表达水平和细胞获得激素非依赖性增殖能力的关系。本研究为下一步拓展性研究打下基础， 丰富了乳腺癌内分泌治疗耐药性产生的理论基础，并为耐药性逆转提供了潜在的分子靶点。